Double-positive Research Articles

Gansui Banxia decoction modulates immune-inflammatory homeostasis to ameliorate malignant ascites in rats

Background“Gansui Banxia decoction” (GBD) is a classical traditional Chinese medicine formula for treating abnormal accumulation of fluid, such as malignant ascites (MA). Although GBD has shown definite water-expelling effects, its exact underlying mechanism has not been elucidated. PurposeThis study aimed to investigate the drug effects of GBD on MA rats and its underlying mechanisms. MethodsThe main chemical composition was determined by ultra-high performance liquid chromatography. The drug effects of GBD was evaluated in the established cancer cell-induced MA rat model. The symptoms were analyzed, and biological samples were collected for detecting immune and inflammation-related indicators by enzyme-linked immunosorbent assays, western blot, and flow cytometry. ResultsGBD increased urine discharge, decreased ascites production, and alleviated cachexia. After GBD treatment, the expression of TLR4, MyD88, and NF-кB and the release of pro-inflammatory cytokines such as interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α were reduced. In addition, GBD increased G1 phase arrest and inhibit excessive proliferation of cells in bone marrow while alleviating G1 phase arrest and increasing proliferation of cells in the thymus. Correspondingly, the development and maturation of T cells also changed. GBD increased the proportion of mature T-cells (CD4+CD8− and CD4−CD8+ single-positive (SP) T-cells), and decrease the proportion of immature cells (CD4+CD8+ double-positive (DP) T-cells and CD4−CD8− double-negative (DN) T-cells) in the blood or tumor microenvironment (TME, the ascites microenvironment). Finally, we further analysis of immune cell subsets, GBD decreased the proportion of immunosuppressive T-cells in the blood (CD4+CD25+Foxp3+T-cells) and TME (CD8+CD25+Foxp3+T-cells), and increased the proportion of anti-tumor immune cells (CD8+CD28+T-cells and NK cells) in the TME. ConclusionThese findings indicated that the drug effects of GBD were attributed to regulating the immune-inflammatory homeostasis, thereby mitigating the destruction of cancer cells and reducing the generation of ascites, which provided theoretical support for the clinical rational application and extended the scientific connotation of “water-expelling” of GBD.

Human HLA-DR+CD27+ regulatory T cells show enhanced antigen-specific suppressive function.

Regulatory T cells (Tregs) have potential for the treatment of autoimmune diseases and graft rejection. Antigen specificity and functional stability are considered critical for their therapeutic efficacy. In this study, expansion of human Tregs in the presence of porcine PBMCs (xenoantigen-expanded Tregs, Xn-Treg) allowed the selection of a distinct Treg subset, coexpressing the activation/memory surface markers HLA-DR and CD27 with enhanced proportion of FOXP3+Helios+ Tregs. Compared with their unsorted and HLA-DR+CD27+ double-positive (DP) cell-depleted Xn-Treg counterparts, HLA-DR+CD27+ DP-enriched Xn-Tregs expressed upregulated Treg function markers CD95 and ICOS with enhanced suppression of xenogeneic but not polyclonal mixed lymphocyte reaction. They also had less Treg-specific demethylation in the region of FOXP3 and were more resistant to conversion to effector cells under inflammatory conditions. Adoptive transfer of porcine islet recipient NOD/SCID IL2 receptor γ-/- mice with HLA-DR+CD27+ DP-enriched Xn-Tregs in a humanized mouse model inhibited porcine islet graft rejection mediated by 25-fold more human effector cells. The prolonged graft survival was associated with enhanced accumulation of FOXP3+ Tregs and upregulated expression of Treg functional genes, IL10 and cytotoxic T lymphocyte antigen 4, but downregulated expression of effector Th1, Th2, and Th17 cytokine genes, within surviving grafts. Collectively, human HLA-DR+CD27+ DP-enriched Xn-Tregs expressed a specific regulatory signature that enabled identification and isolation of antigen-specific and functionally stable Tregs with potential as a Treg-based therapy.

Transient Differentiation-State Plasticity during Initiation of Pediatric Acute Lymphoblastic Leukemia

Introduction Leukemia is characterized by oncogenic lesions that result in a block of differentiation while at the same time intratumor phenotypic plasticity is retained. It is unclear how these two phenomena arise during leukemogenesis in humans. Here we characterized T-cell acute lymphoblastic leukemia (T-ALL), as a model disease to investigate the coherence between leukemia initiating cells (LICs), developmental arrest, and phenotypic plasticity. Methods We characterized 31 primary T-ALL samples for differentiation state heterogeneity using multi parameter flow cytometry. Subsequently, for 5 patients, phenotypic subpopulations were single cell sorted. As leukemic cells do not expand in vitro primary template-directed amplification (PTA) was used to amplify the genome of single blasts to obtain enough DNA for WGS analysis with nucleotide resolution. Shared and unique mutations were used to construct retrospective lineage trees and assess the hierarchy of phenotypically diverse blasts. Results To assess phenotypic differentiation state heterogeneity in T-ALL, we designed a 17-parameter flow cytometry panel to discriminate T-lymphoid developmental cell populations. We characterized 31 samples (median blast count 98% (IQR = 94 - 99)), and defined immunophenotypic subpopulations as populations with a size of >10% of the total CD7+ population. Differentiation state heterogeneity (> 2 subpopulations) was observed in 26 out of the 31 T-ALL patients (83.9%). Interestingly, the most immature CD4-CD8-CD3- “DN-like” differentiation state was found in all samples, suggesting that developmental hierarchy is maintained. We then questioned whether genetic determinants were driving the phenotypic diversity we observed. Therefore 5 T-ALL patients harboring translocations of oncogenes commonly seen in T-ALL were selected, namely, TLX1 (n=1) , LMO2 (n=2) , TAL1 (n=2) and TLX3 (n=1) . For each patient cells of each differentiation state were single cell and bulk sorted for single cell WGS and bulk WGS, respectively. Structural variant analysis confirmed presence of the oncogenic translocation in all single blasts, validating their (pre-)leukemic origin. Moreover, driver analysis showed that type B mutations in e.g., NOTCH1 and PHF6 were shared among all blasts. Interestingly, there were no potential genetic drivers found that were unique to a subpopulation. Neutral passenger mutations were therefore used to construct phylogenetic trees. Herein, blasts with similar phenotypic differentiation states were genetically closer related than blasts of different phenotypes, revealing the heritability of phenotypes (Fig. 1A). The biased distribution of immunophenotypes across the different branches of the phylogenetic tree was then confirmed in bulk WGS samples of sorted phenotypic populations. Suggesting that switching between phenotypes is not a frequent process. Next, the plasticity of T-ALL was assessed by identifying the phenotypic cell state of the LIC. We assessed V(D)J recombination per single blast and identified monoclonal V(D)J recombination for most of the blasts. Moreover, in TAL1 T-ALL we observed monoclonal TRG, TRB and TRA translocation. This suggested that the LIC had undergone complete V(D)J recombination and was a more mature T-cell progenitor. However, blasts with an immature CD4-CD8- (DN) phenotype had undergone the same V(D)J recombination suggesting phenotypic de-differentiation of the LIC. Additionally, in TLX1 T-ALL, the blasts with the more mature CD4+CD8+ double positive (DP) phenotype had monoclonal TRG and TRB gene recombination, but polyclonal TRA recombination, whereas cells with a more immature DN phenotype had no TRA recombination (Fig. 1B). This indicated an immature T-cell progenitor as the LIC and continued differentiation after leukemia initiation resulting in DP cells with unique TRA recombination. Conclusion In conclusion, we show immunophenotypic differentiation state heterogeneity in diagnostic primary T-ALL samples. By coupling phenotypic data with novel single cell whole genome sequencing techniques, we identified heritability of phenotypic cell states. Moreover, we reveal the ability of the LIC to differentiate and de-differentiate after leukemia initiation. Taken together our results demonstrate a transient period of plasticity during leukemia initiation where phenotypic switches appear unidirectional.

CXCR3/CXCL10 Signaling Pathway Drives T-ALL into the CNS/Meningeal Microenvironment

Cure rates for T-cell acute lymphoblastic leukemia (T-ALL) have improved significantly, but central nervous system (CNS) involvement leads to a poor prognosis. Understanding the mechanisms underlying CNS involvement could lead to more effective therapies. The CXCR3/CXCL10 signaling pathway mediates T cell movement across the blood-CSF barrier during neuroinflammation, but its role in T-ALL colonization of the CNS/meninges remains unclear. In our study, we hypothesized that T-ALL cells exploit normal T-cell function to infiltrate the meninges. We examined CXCR3 expression in thymic CD4+CD8+ double positive (DP) cells of ∆E-NOTCH1 T-ALL and control mice. Levels of CXCR3 were higher in CD4 +CD8 + DP cells of T-ALL mice compared to controls. Furthermore, leukemic cells in the meninges, bone marrow (BM), and thymus had higher CXCR3 levels compared to other organs ( p < 0.01), suggesting an organ-specific CXCR3 expression in infiltrating T-ALL cells. Additionally, primary T-ALL samples (n = 24) had elevated CXCR3 mRNA levels compared to normal thymocytes. Using CRISPR-Cas9, we deleted CXCR3 in the KOPTK1 and PER117 cell lines and in two primary patient samples. CXCR3 deletion resulted in decreased proliferation, delayed cell cycle progression in S/G2/M phases but did not affect apoptotic cell death. CXCR3 inactivation led to decreased activation of AKT, ERK1/2, JNK, and reduced the expression of cell motility regulators. Notably, T-ALL cells migrated more efficiently to CXCL10 than to CXCL9 and CXCL11, which was reduced upon CXCR3 silencing ( p < 0.01). Mechanistically, we demonstrated that USP7, a ubiquitin-specific protease, regulates and stabilizes the CXCR3 protein via deubiquitination. To explore the role of CXCR3 in T-ALL in vivo, we transplanted CXCR3 KO and control KOPTK1 cells into NSG mice (8 mice/group). Mice with CXCR3 KO had increased survival and significantly lower leukemia burden in the BM and extramedullary tissues compared to the control group ( p < 0.01). We next analyzed the levels of CXCR3 ligands, CXCL9, CXCL10, and CXCL11, in the blood serum and CSF of ∆E-NOTCH1 and control mice (5 mice/group). T-ALL mice had elevated CXCL10 levels in both blood serum and CSF, with higher levels in the CSF, suggesting compartmentalization of the immune response between the CSF and blood ( p < 0.01). CXCL9 and CXCL11 expression were minimal or undetectable. Remarkably, upon injecting ∆E-NOTCH1-transformed hematopoietic progenitors into CXCL10 knockout ( CXCL10 KO) and control B6 mice (5 mice/group), tissue analysis revealed a significant decrease in T-ALL meningeal infiltration in the CXCL10 KO mice, but not in other organs ( p < 0.01). Conversely, control mice exhibited high levels of leukemic cells in all tested organs. We found that meningeal stromal cells (PDGFR+, CD13+, CD31-, CD45-) expressed CXCL10 in ∆E-NOTCH1 mice, unlike hematopoietic (CD45+) and endothelial cells (CD31+, CD45-), compared to control mice. We further observed upregulation of CXCL10 in human meningeal stromal cells co-cultured with KOPTK1, PER117, and primary samples. Treatment with a CXCR3 antagonist or knockout of CXCR3 reduced leukemic cell migration to the meningeal stromal cells compared to controls ( p < 0.01). Conversely, migration of T-ALL cells to the tested stromal cells was reduced upon treatment with a CXCL10 neutralizing antibody or by CXCL10 deletion in stromal cells. Additionally, we identified that the CXCR3/CXCL10 signaling pathway regulated the expression of VLA-4 and VCAM-1, promoting T-ALL cell adhesion to the meningeal stroma. Genetic inactivation or pharmacological inhibition of CXCR3 in T-ALL cells or CXCL10 in meningeal stromal cells reduced cell-cell adhesion and the expression of VLA-4 and VCAM-1 in T-ALL and meningeal stroma, respectively, further supporting the role of CXCL10-CXCR3 in regulating T-ALL adhesion. Lastly, we investigated whether inflammatory cytokines derived from T-ALL induce CXCL10 in the meningeal microenvironment. We found elevated expression of Ifng, Tnf and Il27 in T-ALL cells infiltrating the meninges, but not other organs of ΔE-NOTCH1 mice ( p < 0.01). Silencing these genes in human T-ALL cells resulted in reduced CXCL10 expression and secretion, along with reduced leukemic cell migration to the tested meningeal stromal cells. In summary, our study emphasizes the key role of CXCR3-CXCL10 signaling in T-ALL progression and meningeal infiltration.

Analysis of characteristics related to interstitial lung disease or pulmonary hypertension in patients with dermatomyositis.

Dermatomyositis (DM) is often associated with interstitial lung disease (ILD) or pulmonary hypertension (PH). The aim of this study was to investigate the clinical characteristics of DM patients with ILD or PH. This study retrospectively analysed the clinical characteristics of 372 patients with DM, including cytokines, lymphocyte subsets, immunoglobulin and complement. The DM patients were divided into different groups according to whether complicated with ILD, PH or anti-melanoma differentiation-associated gene 5 antibodies (MDA5). A qualitative and quantitative data analysis was performed. IgG, IgA and IgM in the DM-associated ILD (ILD-DM) were higher than that of the DM non-complicating ILD (Non-ILD-DM) (p= 0.022, 0.002 and 0.029, respectively). Meanwhile, IL-6 (p= 0.008) and IL-10 (p= 0.001) were increased in the DM-associated PH (PH-DM) than in the DM non-complicating PH (Non-PH-DM), while IL-17 (p= 0.004), double positive (DP) cell ratio and B lymphocyte ratio were reduced in the PH-DM. Moreover, the incidence of ILD and levels of C4 were higher in the DM with MDA5 (MDA5+ DM) than that of the DM without MDA5. ILD-DM has higher IgG, IgA and IgM than that of Non-ILD-DM. PH-DM has higher IL-6, IL-10 and lower IL-17, DP cell ratio and B lymphocyte ratio than that of Non-PH-DM.

Cell-Type Specific Requirement of Glucose Catabolism in Erythropoiesis and Thymopoiesis

Glucose catabolism through glycolysis is central to cellular life. In glycolysis, pyruvate is converted to lactate by lactate dehydrogenase (LDH) or oxidized into the tricarboxylic acid (TCA) cycle via acetyl-CoA by pyruvate dehydrogenase (PDH). However, the two enzymes LDH and PDH required for completing glucose catabolism have never been ablated simultaneously in any tissue in vivo to test if glycolysis is required for cell survival and function. Ldha and Ldhb, the genes coding for LDH have not been ablated simultaneously in in the erythroid lineage to test if LDH plays a significant role in erythropoiesis. It is also unknown if erythoid progenitor cells, which unlike mature erythrocytes contain mitochondria, rely on LDH or are able to oxidize glucose via PDH to support erythroid development. To address these questions, we have generated conditional genetic knockouts (KO) of Ldha, Ldhb and PDH ( Pdha1) in the hematopoietic system to study the requirement of glucose catabolism in erythropoiesis. We observed that Ldha Δ/Δ and Ldha Δ/Δ;Ldhb Δ/Δ but not Ldhb Δ/Δmice had anemia, consistent with an essential role for LDHA in erythrocytes or erythropoiesis. Anemia was more severe in Ldha Δ/Δ;Pdha1 Δ mice as compared to Ldha Δ/Δ mice suggesting LDH and PDH are required in concert to maintain erythroid development in vivo. Ldha Δ/Δ mice accumulated CD71 +Ter119 + erythroid progenitors but Ldha Δ/Δ;Pdha1 Δ knockout mice had a significant decrease in this population. Moreover, there was a more pronounced expansion of upstream CD71 -Ter119 int and CD71 +Ter119 int cells in Ldha Δ/Δ;Pdha1 Δmice. This suggest that the accumulation of CD71 +Ter119 + cells in Ldha Δ/Δ mice is dependent on PDH and that the presence of either LDHA or PDH is required for the progression from CD71 +Ter119 int to CD71 +Ter119 + erythroid progenitors. The bone marrow of Ldha Δ/Δ;Pdha1 Δ showed an accumulation of pre-CFU-E and a depletion of CFU-E cells as compared to single Ldha Δ/Δ mice. Therefore, the presence of either LDHA or PDH is required to transit from pre-CFU-E to CFU-E stage during erythroid differentiation. Our previous results show that PDH is required for the double positive (DP) stage of T cell development. Analysis of thymus of Ldha Δ/Δ and Ldha Δ/Δ;Ldhb Δ/Δ mice did not show any changes in thymocytes. However, Ldha Δ/Δ;Pdha1 Δmice showed a significant decrease at the double negative (DN1) stage which persisted through all T cell development stages. Given that, PDH is required at the double positive (DP) stage and LDH deficiency alone does not affect T cell development, this suggest that LDH and PDH act redundantly at different stages to support T cell development. Altogether, these results suggest cell-type specific usage of glucose catabolic pathways in different differentiation stages. Our results also suggest that progenitor cells can switch between LDH and PDH for glucose catabolism while mature immune cells rely on either LDH or PDH for survival. To investigate this, we will use metabolomics and isotope tracing methods to analyze the route of glucose catabolism during erythroid and T cell development.

Immunological Phenotyping of Mice with a Point Mutation in Cdk4.

Cyclin-dependent kinases (CDKs) play a crucial role in regulation of the mammalian cell cycle. CDK4 and CDK6 control the G1/S restriction checkpoint through their ability to associate with cyclin D proteins in response to growth factor signals. CDK4 deficiency in mice gives rise to a range of endocrine-specific phenotypes including diabetes, infertility, dwarfism, and atrophy of the anterior pituitary. Although CDK6 deficiency can cause thymic atrophy due to a block in the double-negative (DN) to double-positive (DP) stage of T cell development, there are no overt defects in immune cell development reported for CDK4-deficient mice. Here, we examined the impact of a novel N-ethyl-N-nitrosourea-induced point mutation in the gene encoding CDK4 on immune cell development. Mutant mice (Cdk4wnch/wnch) showed normal development and differentiation of major immune cell subsets in the thymus and spleen. Moreover, T cells from Cdk4wnch/wnch mice exhibited normal cytokine production in response to in vitro stimulation. However, analysis of the mixed bone marrow chimeras revealed that Cdk4wnch/wnch-derived T cell subsets and NK cells are at a competitive disadvantage compared to Cdk4+/+-derived cells in the thymus and periphery of recipients. These results suggest a possible role for the CDK4wnch mutation in the development of some immune cells, which only becomes apparent when the Cdk4wnch/wnch mutant cells are in direct competition with wild-type immune cells in the mixed bone marrow chimera.

Legend or Truth: Mature CD4+CD8+ Double-Positive T Cells in the Periphery in Health and Disease.

The expression of CD4 and CD8 co-receptors defines two distinct T cell populations with specialized functions. While CD4+ T cells support and modulate immune responses through different T-helper (Th) and regulatory subtypes, CD8+ T cells eliminate cells that might threaten the organism, for example, virus-infected or tumor cells. However, a paradoxical population of CD4+CD8+ double-positive (DP) T cells challenging this paradigm has been found in the peripheral blood. This subset has been observed in healthy as well as pathological conditions, suggesting unique and well-defined functions. Furthermore, DP T cells express activation markers and exhibit memory-like features, displaying an effector memory (EM) and central memory (CM) phenotype. A subset expressing high CD4 (CD4bright+) and intermediate CD8 (CD8dim+) levels and a population of CD8bright+CD4dim+ T cells have been identified within DP T cells, suggesting that this small subpopulation may be heterogeneous. This review summarizes the current literature on DP T cells in humans in health and diseases. In addition, we point out that strategies to better characterize this minor T cell subset's role in regulating immune responses are necessary.

Characterization of fetal microchimeric immune cells in mouse maternal hearts during physiologic and pathologic pregnancies.

Introduction: During pregnancy, fetal cells can be incorporated into maternal tissues (fetal microchimerism), where they can persist postpartum. Whether these fetal cells are beneficial or detrimental to maternal health is unknown. This study aimed to characterize fetal microchimeric immune cells in the maternal heart during pregnancy and postpartum, and to identify differences in these fetal microchimeric subpopulations between normal and pregnancies complicated by spontaneous preterm induced by ascending infection. Methods: A Cre reporter mouse model, which when mated with wild-type C57BL/6J females resulted in cells and tissues of progeny expressing red fluorescent protein tandem dimer Tomato (mT+), was used to detect fetal microchimeric cells. On embryonic day (E)15, 104 colony-forming units (CFU) E. coli was administered intravaginally to mimic ascending infection, with delivery on or before E18.5 considered as preterm delivery. A subset of pregnant mice was sacrificed at E16 and postpartum day 28 to harvest maternal hearts. Heart tissues were processed for immunofluorescence microscopy and high-dimensional mass cytometry by time-of-flight (CyTOF) using an antibody panel of immune cell markers. Changes in cardiac physiologic parameters were measured up to 60days postpartum via two-dimensional echocardiography. Results: Intravaginal E. coli administration resulted in preterm delivery of live pups in 70% of the cases. mT + expressing cells were detected in maternal uterus and heart, implying that fetal cells can migrate to different maternal compartments. During ascending infection, more fetal antigen-presenting cells (APCs) and less fetal hematopoietic stem cells (HSCs) and fetal double-positive (DP) thymocytes were observed in maternal hearts at E16 compared to normal pregnancy. These HSCs were cleared while DP thymocytes persisted 28days postpartum following an ascending infection. No significant changes in cardiac physiologic parameters were observed postpartum except a trend in lowering the ejection fraction rate in preterm delivered mothers. Conclusion: Both normal pregnancy and ascending infection revealed distinct compositions of fetal microchimeric immune cells within the maternal heart, which could potentially influence the maternal cardiac microenvironment via (1) modulation of cardiac reverse modeling processes by fetal stem cells, and (2) differential responses to recognition of fetal APCs by maternal T cells.

Prediction of Survival in Diffuse Large B Cell Lymphoma According to Immunohistochemistry

BACKGROUND: The introduction of chemoimmunotherapy in the treatment of diffuse large B cell lymphoma (DLBCL) dramatically improved the outcomes compared to chemotherapy alone. However, a significant part of the patients become refractory and eventually relapse. AIM: The purpose of this study is to analyze the survival differences between different groups of DLBCL according to Han’s algorithm. MATERIALS AND METHODS: We will study the medical records of 244 patients treated with RCHOP as first-line therapy who are diagnosed with de novo DLBCL in a cohort of 10 years in the University Clinical Center of Kosovo. According to immunohistochemical markers, the patients will be classified into two major groups, germinal center B cell-like (GCB) and non-germinal center B cell-like (non-GCB) subtypes, and five subgroups (Group 1, Group 2, Group 3 or double positive, Group 4, and Group 5 or triple negative [TN]). RESULTS: The patients in the GCB group have better progression-free survival (PFS) and overall survival (OS) than the non-GCB group. Despite this, double positive (DP) (CD10+MUM1+) and TN (CD10−Bcl6−MUM−) subgroups showed different PFS and OS to the same cell-of-origin group. The DP group showed similar OS and PFS with the non-GCB group, whereas the TN group showed similar OS and PFS with the GCB group. CONCLUSION: These factors will provide valuable knowledge for predicting the prognosis and redirecting the choice of treatment for different groups of DLBCL.

CD1b glycoprotein, a crucial marker of thymocyte development during T cell maturation in cynomolgus monkeys

Phenotypic markers that denote different developmental stages of thymocytes are important for understanding T cell development in the thymus. Here, we show that CD1b is a critical discriminator of thymocyte maturation stage in cynomolgus monkeys. CD1b was expressed by immature thymocytes prior to β-selection, and its expression decreased as cells became fully mature in the thymus. MHC-I expression was lowest at the CD3loCD1b+ immature double-positive (DP) stage, while the ratio of CD1d:MHC-I expression was significantly higher at this stage than at other developmental stages. PLZF was expressed by < 0.2% of thymocytes; most PLZF+ thymocytes were CD3-/loCD1b+ immature DP thymocytes with the potential to produce IL-4. EOMES+ thymocytes, which accounted for > 2% of total thymocytes, were mostly CD3+CD1b- mature thymocytes and predominantly of the CD8 single-positive (SP) lineage. An unconventional CD8+ T cell subset expressing the NKG2AC+CXCR3+ innate-like T cell marker was identified within the EOMES+ CD8 SP lineage; these cells exhibited a memory phenotype. Taken together, these findings show that CD1b is a valuable discriminatory marker of thymocyte development. The data presented herein can be used to characterize the features of PLZF- and EOMES-associated unconventional T cells in the thymus.

Predictive value of CD4+CD8+ double positive T cells for the severity of hemorrhagic fever with renal syndrome

PurposesWe aimed to investigate the levels of CD4+CD8+ double positive (DP) T cells in patients with various severities of hemorrhagic fever with renal syndrome (HFRS), and the predictive capacity of DP T cells for the severity of this disorder. MethodsThe levels of DP T cells in 213 patients and 48 healthy donors were measured by flow cytometry, as were the levels of CD4+ T cells, CD8+ T cells, B lymphocytes, and natural killer (NK) cells. In each type of HFRS patient, we tested the basic clinical reference values for leukocytes, platelets, creatinine (Cr), uric acid (UA), and urea, and the values for activated partial thromboplastin time, prothrombin time, and fibrinogen, using conventional methods. The colloidal gold method was used to measure HFRS antibody levels in the patients. ResultsThe frequency of DP T cells increased with disease severity and peaked in patients with critical disease. Furthermore, the level of DP T cells proportionally correlated with the levels of Cr, UA, and urea in the serum. In contrast, there was an inverse correlation between DP T cells and platelets. Interestingly, the pattern of change in DP T cell frequency was similar to those of CD8+ T cells, B cells, and NK cells, but an inverse tendency was observed for CD4+ T cells. DP T cells demonstrated significant predictive value for the severity of HFRS. ConclusionsThe level of DP T cells is associated with HFRS severity, suggesting that it may be a potent indicator for the course of this disorder.

A stepwise and digital pattern of RSK phosphorylation determines the outcome of thymic selection

Developing CD4+CD8+ double-positive (DP) thymocytes with randomly generated Tcell receptors (TCRs) undergo positive (maturation) or negative (apoptosis) selection on the basis of the strength of TCR stimulation. Selection fate is determined by engagement of TCR ligands with a subtle difference in affinity, but the molecular details of TCR signaling leading to the different selection outcomes have remained unclear. We performed phosphoproteome analysis of DP thymocytes and found that p90 ribosomal protein kinase (RSK) phosphorylation at Thr562 was induced specifically by high-affinity peptide ligands. Such phosphorylation of RSK triggered its translocation to the nucleus, where it phosphorylated the nuclear receptor Nur77 and thereby promoted its mitochondrial translocation for apoptosis induction. Inhibition of RSK activity protected DP thymocytes from antigen-induced cell death. We propose that RSK phosphorylation constitutes a mechanism by which DP thymocytes generate a stepwise and binary signal in response to exposure to TCR ligands with a graded affinity.

High seroprevalence of Leishmania infantum is linked to immune activation in people with HIV: a two-stage cross-sectional study in Bahia, Brazil.

Visceral leishmaniasis is an opportunistic disease in HIV-1 infected individuals, unrecognized as a determining factor for AIDS diagnosis. The growing geographical overlap of HIV-1 and Leishmania infections is an emerging challenge worldwide, as co-infection increases morbidity and mortality for both infections. Here, we determined the prevalence of people living with HIV (PWH) with a previous or ongoing infection by Leishmania infantum and investigated the virological and immunological factors associated with co-infection. We adopted a two-stage cross-sectional cohort (CSC) design (CSC-I, n = 5,346 and CSC-II, n = 317) of treatment-naïve HIV-1-infected individuals in Bahia, Brazil. In CSC-I, samples collected between 1998 and 2013 were used for serological screening for leishmaniasis by an in-house Enzyme-Linked Immunosorbent Assay (ELISA) with SLA (Soluble Leishmania infantum Antigen), resulting in a prevalence of previous or ongoing infection of 16.27%. Next, 317 PWH were prospectively recruited from July 2014 to December 2015 with the collection of sociodemographic and clinical data. Serological validation by two different immunoassays confirmed a prevalence of 15.46 and 8.20% by anti-SLA, and anti-HSP70 serology, respectively, whereas 4.73% were double-positive (DP). Stratification of these 317 individuals in DP and double-negative (DN) revealed a significant reduction of CD4+ counts and CD4+/CD8+ ratios and a tendency of increased viral load in the DP group, as compared to DN. No statistical differences in HIV-1 subtype distribution were observed between the two groups. However, we found a significant increase of CXCL10 (p = 0.0076) and a tendency of increased CXCL9 (p = 0.061) in individuals with DP serology, demonstrating intensified immune activation in this group. These findings were corroborated at the transcriptome level in independent Leishmania- and HIV-1-infected cohorts (Swiss HIV Cohort and Piaui Northeast Brazil Cohort), indicating that CXCL10 transcripts are shared by the IFN-dominated immune activation gene signatures of both pathogens and positively correlated to viral load in untreated PWH. This study demonstrated a high prevalence of PWH with L. infantum seropositivity in Bahia, Brazil, linked to IFN-mediated immune activation and a significant decrease in CD4+ levels. Our results highlight the urgent need to increase awareness and define public health strategies for the management and prevention of HIV-1 and L. infantum co-infection.

Propagation behavior in rectangular metallic waveguide periodically loaded with single negative metamaterial slabs

This paper focused on propagation characteristics of a single mode supporting waveguide enclosed by metallic sidewalls periodically loaded with lossless single negative (SNG) metamaterial slabs along axial direction. The gap between two successive SNG slabs is filled with a double positive (DPS) material. For the analysis, the periodic structure is assumed having symmetric unit cell (UC) and simplified as a two-port network which is one-half of the symmetric UC. Scattering parameters (S-parameters) are defined by even/odd mode analysis of the symmetric UC, and then the total S-parameters are calculated by solving the cascade connection of N unit cells. Eigenvalue equations are also obtained to explicit the solutions of band edge and pass/stopband diagrams in the microwave regime. Since a SNG can be either epsilon-negative (ENG) or mu-negative (MNG), S-parameters and dispersion behavior are illustrated with numerical examples and discussed for each periodic structure constructed with ENG or MNG slabs.

Paxbp1 is indispensable for the survival of CD4 and CD8 double-positive thymocytes.

The lifespan of double-positive (DP) thymocytes is critical for intrathymic development and shaping the peripheral T cell repertoire. However, the molecular mechanisms that control DP thymocyte survival remain poorly understood. Paxbp1 is a conserved nuclear protein that has been reported to play important roles in cell growth and development. Its high expression in T cells suggests a possible role in T cell development. Here, we observed that deletion of Paxbp1 resulted in thymic atrophy in mice lacking Paxbp1 in the early stages of T cell development. Conditional loss of Paxbp1 resulted in fewer CD4+CD8+ DP T cells, CD4 and CD8 single positive (SP) T cells in the thymus, and fewer T cells in the periphery. Meanwhile, Paxbp1 deficiency had limited effects on the CD4-CD8- double negative (DN) or immature single-positive (ISP) cell populations. Instead, we observed a significant increase in the susceptibility of Paxbp1-deficient DP thymocytes to apoptosis. Consistent with this, RNA-Seq analysis revealed a significant enrichment of the apoptotic pathway within differentially expressed genes in Paxbp1-deficient DP cells compared to control DP cells. Together, our results suggest a new function for Paxbp1, which is an important mediator of DP thymocyte survival and critical for proper thymic development.

Scattering of Laguerre–Gaussian Beam by a Topological Insulator Cylinder Coated with Metamaterials

A theoretical investigation is presented to study the orbital angular momentum (OAM) beam scattering from a topological insulator (TI) cylinder coated with metamaterials. To calculate the electromagnetic (EM) response of the scattered field, the field equations are modified using Laguerre–Gaussian (LG) potential and plane wave scattering. The response of the metamaterial coated geometry has been reported for Double positive (DPS), Double negative (DNG), Epsilon negative (ENG), and Mu negative (MNG) coating layers, respectively. The normalized bistatic echo widths for the TI cylinder also change by changing the topological charge ( $$l$$ ), the radius of the metamaterial coated layer $$(b)$$ , source frequency $$(f)$$ , and beam waist radius $$({w}_{0})$$ . Our result for $${LG}_{00}$$ beam exactly tones with plane wave scattering as a reference. Numerical results in the form of bistatic echo widths have been calculated. Besides, the prementioned parameters, the influence of OAM mode index on the bistatic echo width is also analyzed. It can be seen that OAM mode number plays major role in controlling the scattering amplitude and intensity.

Tox4 regulates transcriptional elongation and reinitiation during murine T cell development

HMG protein Tox4 is a regulator of PP1 phosphatases with unknown function in development. Here we show that Tox4 conditional knockout in mice reduces thymic cellularity, partially blocks T cell development, and decreases ratio of CD8 to CD4 through decreasing proliferation and increasing apoptosis of CD8 cells. In addition, single-cell RNA-seq discovered that Tox4 loss also impairs proliferation of the fast-proliferating double positive (DP) blast population within DP cells in part due to downregulation of genes critical for proliferation, notably Cdk1. Moreover, genes with high and low expression level are more dependent on Tox4 than genes with medium expression level. Mechanistically, Tox4 may facilitate transcriptional reinitiation and restrict elongation in a dephosphorylation-dependent manner, a mechanism that is conserved between mouse and human. These results provide insights into the role of TOX4 in development and establish it as an evolutionarily conserved regulator of transcriptional elongation and reinitiation.

The Wiskott-Aldrich syndrome protein is required for positive selection during T-cell lineage differentiation.

The Wiskott-Aldrich syndrome (WAS) is an X-linked primary immune deficiency caused by a mutation in the WAS gene. This leads to altered or absent WAS protein (WASp) expression and function resulting in thrombocytopenia, eczema, recurrent infections, and autoimmunity. In T cells, WASp is required for immune synapse formation. Patients with WAS show reduced numbers of peripheral blood T lymphocytes and an altered T-cell receptor repertoire. In vitro, their peripheral T cells show decreased proliferation and cytokine production upon aCD3/aCD28 stimulation. It is unclear whether these T-cell defects are acquired during peripheral activation or are, in part, generated during thymic development. Here, we assessed the role of WASp during T-cell differentiation using artificial thymic organoid cultures and in the thymus of humanized mice. Although CRISPR/Cas9 WAS knockout hematopoietic stem and progenitor cells (HSPCs) rearranged the T-cell receptor and differentiated to T-cell receptor (TCR)+ CD4+ CD8+ double-positive (DP) cells similar to wild-type HSPCs, a partial defect in the generation of CD8 single-positive (SP) cells was observed, suggesting that WASp is involved in their positive selection. TCR repertoire analysis of the DP and CD8+ SP population, however, showed a polyclonal repertoire with no bias toward autoreactivity. To our knowledge, this is the first study of the role of WASp in human T-cell differentiation and on TCR repertoire generation.

Mettl3-m6A-Creb1 forms an intrinsic regulatory axis in maintaining iNKT cell pool and functional differentiation

N6-methyladenosine (m6A) methyltransferase Mettl3 is involved in conventional Tcell immunity; however, its role in innate immune cells remains largely unknown. Here, we show that Mettl3 intrinsically regulates invariant natural killer T (iNKT) cell development and function in an m6A-dependent manner. Conditional ablation of Mettl3 in CD4+CD8+ double-positive (DP) thymocytes impairs iNKT cell proliferation, differentiation, and cytokine secretion, which synergistically causes defects in B16F10 melanoma resistance. Transcriptomic and epi-transcriptomic analyses reveal that Mettl3 deficiency disturbs the expression of iNKT cell-related genes with altered m6A modification. Strikingly, Mettl3 modulates the stability of the Creb1 transcript, which in turn controls the protein and phosphorylation levels of Creb1. Furthermore, conditional targeting of Creb1 in DP thymocytes results in similar phenotypes of iNKT cells lacking Mettl3. Importantly, ectopic expression of Creb1 largely rectifies such developmental defects in Mettl3-deficient iNKT cells. These findings reveal that the Mettl3-m6A-Creb1 axis plays critical roles in regulating iNKT cells at the post-transcriptional layer.